Microfloral biomass having omega-3 highly unsaturated fatty acids

ABSTRACT

Disclosed is a process for growing the microflora Thraustochytrium, Schizochytrium, and mixtures thereof, which includes the growing of the microflora in fermentation medium containing non-chloride containing sodium salts, in particular sodium sulfate. In a preferred embodiment of the present invention, the process produces microflora having a cell aggregate size of less than about 150 microns and a high content of omega-3 highly unsaturated fatty acids useful for the production of food products for use in aquaculture. Further disclosed is a food product which includes Thraustochytrium, Schizochytrium, and mixtures thereof, and a component selected from flaxseed, rapeseed, soybean and avocado meal. Such a food product includes a balance of long chain and short chain omega-3 highly unsaturated fatty acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.07/962522, filed Oct. 16, 1992, now U.S. Pat. No. 5,340,742 which is acontinuation-in-part of copending and commonly assigned U.S. patentapplication Ser. No. 07/911,760, filed Jul. 10, 1992 now U.S. Pat. No.5,340,594 which is incorporated herein in its entirety by referencewhich is a divisional of U.S. patent application Ser. No. 07/580,778,filed Sep. 11, 1990 which is incorporated herein in its entirety whichissued as U.S. Pat. No. 5,130,242 which is a continuation-in-part of andcommonly assigned U.S. patent application Ser. No. 07/439,093, filedNov. 17, 1989, now abandoned, which is incorporated herein in itsentirety by reference which is a continuation-in-part of U.S. patentapplication Ser. No. 07/241,410, filed Sep. 7, 1988, now abandoned,which was previously expressly abandoned.

FIELD OF THE INVENTION

The field of this invention relates to heterotrophic organisms and aprocess for culturing them for the production of lipids with highconcentrations of omega-3 highly unsaturated fatty acids (HUFA) suitablefor human and animal consumption as food additives or for use inpharmaceutical and industrial products.

BACKGROUND OF THE INVENTION

Omega-3 highly unsaturated fatty acids (HUFAs) are of significantcommercial interest in that they have been recently recognized asimportant dietary compounds for preventing arteriosclerosis and coronaryheart disease, for alleviating inflammatory conditions and for retardingthe growth of tumor cells. These beneficial effects are a result both ofomega-3 HUFAs causing competitive inhibition of compounds produced fromomega-6 fatty acids, and from beneficial compounds produced directlyfrom the omega-3 HUFAs themselves (Simopoulos et al., 1986). Omega-6fatty acids are the predominant HUFAs found in plants and animals.Currently, a commercially available dietary source of omega-3 HUFAs isfrom certain fish oils which can contain up to 20-30% of these fattyacids. The beneficial effects of these fatty acids can be obtained byeating fish several times a week or by daily intake of concentrated fishoil. Consequently large quantities of fish oil are processed andencapsulated each year for sale as a dietary supplement. However, thereare several significant problems with these fish oil supplements,including bioaccumulation of fat-soluble vitamins and high levels ofsaturated and omega-6 fatty acids, both of which can have deleterioushealth effects.

Another source of omega-3 HUFAS is the microflora Thraustochytrium andSchizochytrium which are discussed in detail in related U.S. Pat. No.5,130,242. These microflora have the advantages of being heterotrophicand capable of high levels of omega-3 HUFA production. There stillexists a need however for improved methods for fermentation of thesemicroflora and identification of improved uses of the microfloraproduct.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a new process for growing themicroflora Thraustochytrium, Schizochytrium, and mixtures thereof, whichincludes the growing of the microflora in a culture medium containingnon-chloride containing sodium salts, particularly including sodiumsulfate. More particularly, a significant portion of the sodiumrequirements of the fermentation are supplied as a non-chloridecontaining sodium salt. The present process is particularly useful incommercial production because the chloride content in the medium can besignificantly reduced, thereby avoiding the corrosive effects ofchloride on fermentation equipment. In addition, the present inventionis particularly useful for production of food products for use inaquaculture because Thraustochytrium and Schizochytrium cultured in suchmedia form much smaller clumps than those cultured in high chloridemedia and are thus more available as a food source for larval shrimp. Inparticular, Thraustochytrium and Schizochytrium cultured in mediumcontaining sodium sulfate can have cell aggregates of an average size ofless than about 150 microns in diameter.

A further embodiment of the present invention is the production of amicroflora biomass comprising Thraustochytrium, Schizochytrium, andmixtures thereof which have an average cell aggregate size of less thanabout 150 microns. The microflora biomass is useful for aquaculture andin particular, for feeding larval shrimp because the microflora have theprimary feed advantages required for shrimp of a high sterol content anda high omega-3 highly unsaturated fatty acid (HUFA) content.Additionally, because of the small cell aggregate size, the microfloracan be eaten by the larval shrimp, brine shrimp, rotifers, and mollusks.The present invention further includes a process for the production ofthese organisms which includes feeding Thraustochytrium, Schizochytrium,and mixtures thereof, having an average cell size of less than about 150microns to them.

A further embodiment of the present invention is directed to a foodproduct which is comprised of microflora selected from the groupconsisting of Thraustochytrium, Schizochytrium, and mixtures thereof andan additional component selected from the group consisting of flaxseed,rapeseed, soybean, avocado meal, and mixtures thereof. A particularadvantage of this food product is that it has a high long chain omega-3fatty acid content and a high short chain omega-3 fatty chain contentfrom the additional component. In a further embodiment, the food productis produced by extrusion. The extrusion process involves mixing themicroflora with the additional component, thereby reducing the moisturecontent of the food product. The food product is then extruded underheat, thus driving off a significant portion of the reduced moisture.The remaining amount of the original moisture content is readily removedby air drying or short baking times, thereby reducing the overall energyrequirements of drying and the potential degradation of the omega-3HUFA's by extended drying at high temperatures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of HUFA production in newlyisolated strains of the invention, represented by , and previouslyisolated strains represented by +. Each point represents a strain, theposition of each point is determined by the percent by weight of totalfatty acids which were omega-3 HUFAs (abscissa) and the percent byweight of total fatty acids which were omega-6 fatty acids (ordinate).Only those strains of the invention were plotted wherein less than 10.6%(w/w) of total fatty acids were omega-6 and more than 67% of total fattyacids were omega-3.

FIG. 2 is a graphical representation of HUFA production in newlyisolated strains of the invention, represented by , and previouslyisolated strains, represented by +. Each point represents a strain, theposition of each point is determined by the percent by weight of totalfatty acids which were omega-3 HUFAs (abscissa) and percent of weight oftotal fatty acids which were eicosapentaenoic acid (EPA C20:5n-3)(ordinate). Only those strains of the invention were plotted whereinmore than 67% (w/w) of total fatty acids were omega-3 and more than 7.8%(w/w) of total fatty acids were C20:5n-3.

FIG. 3 is a graphical representation of omega-3 HUFA composition innewly isolated strains of the invention, represented by □, andpreviously isolated strains, represented by +. Each point represents aseparate strain. Values on the abscissa are weight fraction of totalomega-3 HUFAs which were C20:5n-3 and on the ordinate are weightfraction of total omega-3 fatty highly unsaturated acids which wereC22:6n-3. Only strains of the invention were plotted having either aweight fraction of C20:5n-3 28% or greater, or a weight fraction ofC22:6n-3 greater than 93.6%.

FIG. 4 is a graph showing growth of various newly isolated strains ofthe invention and previously isolated strains, at 25° C. and at 30° C.Growth rates are normalized to the growth rate of strain U-30 at 25° C.Previously isolated strains are designated by their ATCC accessionnumbers.

FIG. 5 is a graph of total yields of cellular production after inductionby nitrogen limitation. Each of ash-free dry weight, total fatty acidsand omega-3 HUFAs, as indicated, was plotted, normalized to thecorresponding value for strain 28211. All strains are identified by ATCCaccession numbers.

FIG. 6 is a graph of fatty acid yields after growth in culture mediahaving the salinity indicated on the abscissa. Strains shown are newlyisolated strains S31 (ATCC 20888) (□) and U42-2 (ATCC 20891) (+) andpreviously isolated strains, ATCC 28211 ( ) and ATCC 28209 (Δ). Fattyacid yields are plotted as relative yields normalized to an arbitraryvalue of 1.00 based on the average growth rate exhibited by S31 (ATCC20888) (□) over the tested salinity range.

FIG. 7 is a graph of increases in the omega-3 HUFA content of the totallipids in the brine shrimp, Artemia salina, fed Thraustochytrid strain(ATCC 20890) isolated by the method in Example 1. EPA=C20:5n-3; DHA=C22:5n-3.

FIG. 8 is a graph of increases in the omega-3 HUFA content of the totallipids in the brine shrimp, Artemia salina, fed Thraustochytrid strain(ATCC 20888) isolated by the method in Example 1. EPA =C20:5n-3;DHA=C22:5n-3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

For purposes of definition throughout the application, it is understoodherein that a fatty acid is an aliphatic monocarboxylic acid. Lipids areunderstood to be fats or oils including the glyceride esters of fattyacids along with associated phosphatides, sterols, alcohols,hydrocarbons, ketones, and related compounds.

A commonly employed shorthand system is used in this specification todenote the structure of the fatty acids (e.g., Weete, 1980). This systemuses the letter "C" accompanied by a number denoting the number ofcarbons in the hydrocarbon chain, followed by a colon and a numberindicating the number of double bonds, i.e., C20:5, eicosapentaenoicacid. Fatty acids are numbered starting at the carboxy carbon. Positionof the double bonds is indicated by adding the Greek letter delta (Δ)followed by the carbon number of the double bond; i.e.,C20:5omega-3Δ⁵,8,11,14,17. The "omega" notation is a shorthand systemfor unsaturated fatty acids whereby numbering from the carboxy-terminalcarbon is used. For convenience, n-3 will be used to symbolize "omega-3"especially when using the numerical shorthand nomenclature describedherein. Omega-3 highly unsaturated fatty acids are understood to bepolyethylenic fatty acids in which the ultimate ethylenic bond is 3carbons from and including the terminal methyl group of the fatty acid.Thus, the complete nomenclature for eicosapentaenoic acid, an omega-3highly unsaturated fatty acid, would be C20:5n-3Δ⁵,8,11,14,17. For thesake of brevity, the double bond locations (Δ⁵,8,11,14,17) will beomitted. Eicosapentaenoic acid is then designated C20:5n-3,Docosapentaenoic acid (C22:5n3Δ⁷,10,13,16,19) is C22:5n-3, andDocosahexaenoic acid (C22:6n-3Δ⁴,7,10,13,16,19) is C22:6n-3. Thenomenclature "highly unsaturated fatty acid" means a fatty acid with 4or more double bonds. "Saturated fatty acid" means a fatty acid with 1to 3 double bonds.

A collection and screening process has been developed to readily isolatemany strains of microorganisms with the following combination ofeconomically desirable characteristics for the production of omega-3HUFAs: 1) capable of heterotrophic growth; 2) high content of omega-3HUFAs; 3) unicellular; 4) preferably low content of saturated andomega-6 HUFAs; 5) preferably nonpigmented, white or essentiallycolorless cells; 6) preferably thermotolerant (ability to grow attemperatures above 30° C.); and 7) preferably euryhaline (able to growover a wide range of salinities, but especially at low salinities). Thisprocess is described in detail in related U.S. Pat. No. 5,130,242.

Using the collection and screening process, strains of unicellularmicroflora can be isolated which have fatty acid contents up to about45% total cellular dry weight percent (% dwt), and which exhibit growthover a temperature range from 15°-48° C. and grow in a very low salinityculture medium. Many of the very high omega-3 strains are very slowgrowers. Strains which have been isolated by the method outlined above,and which exhibit rapid growth, good production and high omega-3 HUFAcontent, have omega-3 unsaturated fatty acid contents up toapproximately 12% dwt.

One aspect of the present invention is the growth of Thraustochytrium,Schizochytrium, and mixtures thereof with high omega-3 HUFA content, infermentation medium containing non-chloride containing sodium salts andpreferably sodium sulfate. More particularly, a significant portion ofthe sodium requirements of the fermentation are supplied as nonchloridecontaining sodium salts. For example, less than about 75% of the sodiumin the fermentation medium is supplied as sodium chloride, morepreferably less than about 50% and more preferably less than about 25%.A particular advantage of the present invention is that the mediumprovides the source of sodium needed by the microflora to grow in theabsence of a significant amount of chloride which can corrode the vesselin which the microflora are being grown and other fermentation ordownstream processing equipment. It has been surprisingly found thatmicroflora of the present invention can be grown at chlorideconcentrations of less than about 3 gl/l more preferably less than about500 mg/l, more preferably less than about 250 mg/l and more preferablybetween about 60 mg/l and about 120 mg/l while still attaining highyields of biomass per sugar of about 50% or greater. As discussed below,an additional advantage of the present invention is the production ofmicroflora that are high in omega-3 HUFA content but have a small enoughcell aggregate size to be consumed by larval shrimp, brine shrimp,rotifers and mollusks.

Non-chloride containing sodium salts can include soda ash (a mixture ofsodium carbonate and sodium oxide), sodium carbonate, sodiumbicarbonate, sodium sulfate and mixtures thereof, and preferably includesodium sulfate. Soda ash, sodium carbonate and sodium bicarbonate tendto increase the pH of the fermentation medium, thus requiring controlsteps to maintain the proper pH of the medium. The concentration ofsodium sulfate is effective to meet the salinity requirements of themicroflora, preferably the sodium concentration is (expressed as g/l ofNa) is greater than about 1.0 g/l, more preferably between about 1.0 gland about 50.0 g/l and more preferably between about 2.0 g/l and about25 g/l.

It has been surprisingly found that fermentation of the strains in thepresence of a non-chloride containing sodium salt and particularly,sodium sulfate limits the cell aggregate size of the strains to lessthan about 150 microns, preferably less than about 100 microns, and morepreferably less than about 50 microns. As used herein, the term cellaggregate size refers to the approximate average diameter of clumps oraggregates of cells in a fermentation medium of a microfloral culture.Typically, greater than about 25 percent of the cell aggregates in amicrofloral culture have cell aggregate size below the average size,more preferably greater than about 50 percent and more preferablygreater than about 75 percent. Microfloral cells produced in accordancewith the present invention meet cell aggregate size parameters describedabove while in fermentation medium as well as after freezing and/ordrying of the biomass if resuspended in liquid or physically agitated,such as by a blender or vortexer. The present process is particularlyimportant for microflora which replicate by successive bipartition(wherein a single cell replicates by dividing into two cells which eachdivide into two more, etc.) because as cells repeatedly and rapidlyundergo this process, the cells tend to clump forming multi-cellaggregates which are often outside the cell aggregate size parametersidentified above. Schizochytrium replicate by successive bipartition andby forming sporangia which release zoospores. Thraustochytrium, however,replicate only by forming sporangia and releasing zoospores. ForThraustochytrium which replicate by sporangia/zoospore formation,clumping can be a problem as well, particularly because even though thenumber of cells in an aggregate may not be as great as aggregates formedby successive bipartition, the individual cell sizes of Thraustochytriumtend to be larger, and thus, clumps of a small number of cells arelarger. However, one deposited strain of Thraustochytrium, ATCC 26185,has been identified which does not exhibit significant aggregation.

In another aspect of the present invention, it has been found that byrestricting the oxygen content of the fermentation medium during thegrowth of Thraustochytrium, Schizochytrium, and mixtures thereof, thelipid content of the strains can be increased. The optimum oxygenconcentration for lipid production can be determined for any particularmicroflora by variation of the oxygen content of the medium. Inparticular, the oxygen content of the fermentation medium is maintainedat an oxygen content of less than about 40% of saturation and preferablybetween about 5% of saturation and about 40% of saturation.

Growth of the strains by the invention process can be effected at anytemperature conducive to satisfactory growth of the strains; forexample, between about 5° C. and about 48° C., preferably between about15° C. and about 40° C., and more preferably between about 25° C. andabout 35° C. The culture medium typically becomes more alkaline duringthe fermentation if pH is not controlled by acid addition or buffers.The strains will grow over a pH range from 5.0-11.0 with a preferablerange of about 6.0-8.5.

Various fermentation parameters for inoculating, growing and recoveringmicroflora are discussed in detail in U.S. Pat. No. 5,130,242. Thebiomass harvested from a fermentation run can be dried (e.g., spraydrying, tunnel drying, vacuum drying, or a similar process) and used asa feed or food supplement for any animal whose meat or products areconsumed by humans. Similarly, extracted omega-3 HUFAs can be used as afeed or food supplement. Alternatively, the harvested and washed biomasscan be used directly (without drying) as a feed supplement. To extendits shelf life, the wet biomass can be acidified (approximatepH=3.5-4.5) and/or pasteurized or flash heated to inactivate enzymes andthen canned, bottled or packaged under a vacuum or non-oxidizingatmosphere (e.g., N₂ or CO₂). The term "animal means any organismbelonging to the kingdom Animalia and includes, without limitation, anyanimal from which poultry meat, seafood, beef, pork or lamb is derived.Seafood is derived from, without limitation, fish, shrimp and shellfish.The term "products" includes any product other than meat derived fromsuch animals, including, without limitation, eggs or other products.When fed to such animals, omega-3 HUFAs in the harvested biomass orextracted omega-3 HUFAs are incorporated into the flesh, eggs or otherproducts of such animals to increase the omega-3 HUFA content thereof.

A further embodiment of the present invention is the use of theharvested biomass as a food product for larval shrimp, brine shrimp,rotifers and mollusks and in particular, larval shrimp. During thelarval stage of development, shrimp larvae are unable to use some foodsources because the food source is too large. In particular, at certainstages of development, shrimp larvae are unable to use a food sourcehaving a diameter greater than about 150 microns. Thus, microflora grownin fermentation medium containing a non-chloride sodium salt, andparticularly sodium sulfate, as broadly discussed above, are suitablefor use as a shrimp food product. As discussed above, microflora grownunder such conditions typically have a cell aggregate size less thanabout 150 microns, preferably less than about 100 microns, and morepreferably less than about 50 microns.

A further advantage of the use of microflora of the present invention asa food source for shrimp is that such microflora have a significantsterol content including cholesterol, which is a primary feedrequirement for shrimp. The microflora of the present inventiontypically have a sterol content of preferably at least about 0.1%ash-free dry weight (afdw), more preferably at least about 0.5% afdw,and even more preferably at least about 1.0% afdw. In addition, themicroflora of the present invention typically have a cholesterol contentof preferably at least about 15% of the total sterol content, morepreferably at least about 25% of the total sterol content, and even morepreferably at least about 40% of the total sterol content. Further, themicrofloral biomass of the present invention also provide shrimp withadditional nutritional requirements such as omega-6 fatty acids,protein, carbohydrates, pigments and vitamins.

The microbial product of the present invention is of value as a sourceof omega-3 HUFAs for fish, shrimp and other products produced byaquaculture. The product can be used as a food product as describedabove for shrimp; or added directly as a supplement to the feed forshrimp and fish, generally; or it can be fed to brine shrimp or otherlive feed organisms intended for consumption by an aquaculturedorganism. The use of such microflora in this manner enables the shrimpfarmer to obtain significantly higher growth rates and/or survival ratesfor larval shrimp and to produce post-larval shrimp which are more hardyand robust.

For most feed applications, the fatty acid content of the harvestedcells will be approximately 15-50% dwt with the remaining material beinglargely protein and carbohydrate. The protein can contributesignificantly to the nutritional value of the cells as several of thestrains that have been evaluated have all of the essential amino acidsand would be considered a nutritionally balanced protein.

A further embodiment of the present invention is the production of afood product using the Thraustochytrium, Schizochytrium, and mixturesthereof, of the present invention, combined with an additional componentselected from the group consisting of rapeseed, flaxseed, soybean andavocado meal. A particular advantage of this embodiment is that the foodproduct contains both short chain omega-3 HUFAs from the additionalcomponent and long chain omega-3 HUFAs from the microflora. Foodproducts having flaxseed, rapeseed, soybeans and avocado meal are knownto be useful for supplying a source of short chain omega-3 HUFAs and foradditionally supplying a source of short chain omega-3 HUFAs, which canbe elongated by the humans and animals that ingest them. Such foodproducts, however, have the disadvantages of having high cholinecontents from the additional component, which can form primary aminesand result in an unpleasant fish smell; and toxic compounds from theadditional component, which at high levels can, for example, inhibit thelaying of eggs by hens or cause animals to go off of their feed. Assuch, the food product of the present invention has the advantage of alowered flaxseed, rapeseed, soy bean or avocado meal content because theorganism ingesting the food product does not need high levels of shortchain omega-3 HUFAs for the purpose of converting them to long chainHUFAs. Thus, the lowered content of the flaxseed and rapeseed of thefood product results in lowered amounts of choline and/or inhibitorytoxic compounds present in the food product.

The amount of Thraustochytrium, Schizochytrium, and mixtures thereof,used in the food product can range from between about 5% to about 95% byweight. The additional component can be present in the food product at arange of between about 5% to about 95% by weight. Additionally, the foodproduct can include other components as well, including qrains,supplements, vitamins, binders and preservatives.

In a preferred embodiment, the above food product is produced using anextrusion process. The extrusion process involves mixing the microflorawith the additional component, thereby reducing the moisture in themicrofloral biomass by the amount of the additional component mixed. Thefood product is extruded under heat, thus removing further moisture fromthe food product. The resulting product which has a low moisture contentcan be air dried or dried by relatively short baking times therebyreducing the overall energy requirements of drying and the potentialdegradation of omega-3 HUFAs due to long time periods at hightemperatures. In addition, heat from the extrusion process can degradesome of the unwanted toxic compounds commonly found in the additionalcomponent which can, for example, inhibit egg laying by hens or causeanimals to go off of their feed.

The present invention will be described in more detail by way of workingexamples. Species meeting the selection criteria described above havenot been described in the prior art. By employing these selectioncriteria, over 25 potentially promising strains have been isolated fromapproximately 1000 samples screened. Out of the approximate 20,500strains in the American Type Culture Collection (ATCC), 10 strains werelater identified as belonging to the same taxonomic group as the strainsisolated. Those strains still viable in the Collection were procured andused to compare with strains isolated and cultured by the disclosedprocedures. The results of this comparison are presented in Examples 4and 5 below.

The most recent taxonomic theorists place Thraustochydrids with thealgae or algae-like protists. All of the strains of unicellularmicroorganisms disclosed and claimed herein are members of the orderThraustochytriales (Order: Thraustochytriales; Family:Thraustochytriaceae; Genus: Thraustochytrium or Schizochytrium). Forgeneral purposes of discussion herein, these microorganisms will becalled microflora to better denote their uncertain exact taxonomicposition.

The novel strains identified below were deposited under the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Pat. Procedure. All restrictions on the availabilityto the public of the materials so deposited will be irrevocably removedupon the granting of a patent. Each deposit will be stored for a periodof at least five years after the most recent request for the furnishingof a sample of the deposited microorganism is received by the AmericanType Culture Collection (ATCC), and, in any case, for a period of atleast 30 years after the date of the deposit.

Preferred microorganisms of the present invention have all of theidentifying characteristics of the deposited strains and, in particular,the identifying characteristics of being able to produce omega-3 HUFAsas described herein and having cell aggregate size characteristics whencultured under conditions as described herein. In particular, thepreferred microorganisms of the present invention refer to the followingdeposited microorganisms and mutants thereof.

    ______________________________________                                        Strain          ATCC No.  Deposit Date                                        ______________________________________                                        Schizochytrium S31                                                                            20888     8/8/88                                              Schizochytrium S8                                                                             20889     8/8/88                                              ______________________________________                                    

The present invention, while disclosed in terms of specific organismstrains, is intended to include all such methods and strains obtainableand useful according to the teachings disclosed herein, including allsuch substitutions, modification, and optimizations as would beavailable expedients to those of ordinary skill in the art.

The following examples and test results are provided for the purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1

Collection and Screening

A 150 ml water sample was collected from a shallow, inland saline pondand stored in a sterile polyethylene bottle. Special effort was made toinclude some of the living plant material and naturally occurringdetritus (decaying plant and animal matter) along with the water sample.The sample was placed on ice until return to the laboratory. In the lab,the water sample was shaken for 15-30 seconds, and 1-10 ml of the samplewas pipetted or poured into a filter unit containing 2 types offilters: 1) on top, a sterile 47 mm diameter Whatman #4 filter having apore size about 25 μm; and 2) underneath the Whatman filter, a 47 mmdiameter polycarbonate filter with about 1.0 μm pore size. Given slightvariations of nominal pore sizes for the filters, the cells collected onthe polycarbonate filter range in size from about 1.0 μm to about 25 μm.

The Whatman filter was removed and discarded. The polycarbonate filterwas placed on solid F-1 media in a petri plate, said media consisting of(per liter): 600 ml seawater (artificial seawater can be used), 400 mldistilled water, 10 g agar, 1 g glucose, 1 g protein hydrolysate, 0.2 gyeast extract, 2 ml 0.1M KH₂ PO₄, 1 ml of a vitamin solution (A-vits)(Containing 100 mg/l thiamine, 0.5 mg/l biotin, and 0.5 mg/1cyanocobalamin), 5 ml of a trace metal mixture (PII metals, containingper liter: 6.0 g Na₂ EDTA, 0.29 g FeCl₃ 6H₂ O, 6.84 g H₃ BO₃, 0.86 MnCl₂4H₂ O, 0.06 g ZnCl₂, 0.026 g CoCl₂ 6H₂ O, (0.052 g NiSO₄ H₂ O, 0.002 gCuSo₄ 5H₂ O, and 0.005 g Na₂ MoO₄ 2H₂ O, and 500 mg each of streptomycinsulfate and penicillin-G. The agar plate was incubated in the dark at30° C. After 2-4 days numerous colonies appeared on the filter. Coloniesof unicellular microflora (except yeast) were picked from the plate andrestreaked on a new plate of similar media composition. Specialattention was made to pick all colonies consisting of colorless whitecells. The new plate was incubated at 30° C. and single colonies pickedafter a 2-4 day incubation period. Single colonies were then picked andplaced in 50 ml of liquid medium containing the same organic enrichmentsas in the agar plates. These cultures were incubated for 2-4 days at 30°C. on a rotary shaker table (100-200 rpm). When the cultures appeared toreach maximal density, 20-40 ml of the culture was harvested,centrifuged and lyophilized. The sample was then analyzed by standard,well-known gas chromatographic techniques (e.g., Lepage and Roy, 1984)to identify the fatty acid content of the strain. Those strains withomega-3 HUFAs were thereby identified, and cultures of these strainswere maintained for further screening.

Using the collection and screening process outlined above, over 150strains of unicellular microflora have been isolated which have highomega-3 HUFA contents as a percent of total fatty acids and whichexhibit growth over a temperature range from 15°-48° C. Strains can alsobe isolated which have less than 1% (as % of total fatty acids) of theundesirable C20:4n-6 and C22:5n-6 HUFAs for some applications. Strainswith high omega-6 content can also be isolated. Strains of thesemicroflora can be repeatedly isolated from the same location using theprocedure outlined above. A few of the newly isolated strains have verysimilar fatty acid profiles. The possibility that some are duplicateisolates of the same strain cannot be ruled out at present. Furtherscreening for other desirable traits such as salinity tolerance orability to use a variety of carbon and nitrogen sources can then becarried out using a similar process.

Example 2

Maintaining Unrestricted Growth: PO₄ and Yeast Extract

Cells of Schizochytrium aggregatum (ATCC 28209) were picked from solidF-1 medium and inoculated into 50 ml of FFM medium. (Fuller et al.,1964). This medium contains: seawater, 1000 ml; glucose, 1.0 g; gelatinhydrolysate, 1.0 g; liver extract, 0.01 g; yeast extract, 0.1 g; PIImetals, 5 ml; 1 ml B-vitamins solution (Goldstein et al., 1969); and 1ml of an antibiotic solution (25 g/l streptomycin sulfate andpenicillin-G). 1.0 ml of the vitamin mix (pH 7.2) contains: thiamineHC1,200 μg; biotin, 0.5 μg; cyanocobalamin, 0.05 μg; nicotinic acid, 100μg; calcium pantothenate, 100 μg; riboflavin, 5 0 μg; pyridoxine HC1,40.0 μg; pyridoxamine 2HCl, 20.0 μg; p-aminobenzoic acid, 10 μg;chlorine HC1, 500 μg; inositol, 1.0 mg; thymine, 0.8 mg; orotic acid,0.26 mg; folinic acid, 0.2 μg; and folic acid, 2.5 μg. The culture wasplaced on a rotary shaker (200 rpm) at 27° C. After 3-4 days, 1 ml ofthis culture was transferred to 50 ml of each of the followingtreatments: 1) FFM medium (as control); and 2) FFM medium with theaddition of 250 mg/l KH₂ PO₄ and 250 mg/l yeast extract. These cultureswere placed on a rotary shaker (200 rpm) at 27° C. for 48 hr. The cellswere harvested and the yield of cells quantified. In treatment 1, thefinal concentration of cells on an ash-free dry weight basis was 616mg/l. In treatment 2, the final concentration of cells was 1675mg/l,demonstrating the enhanced effect of increasing PO₄ and yeastextract concentrations in the culture medium.

Example 3

Maintaining Unrestricted Growth: Substitution of Corn Steep. Liquor forYeast Extract

Cells of Schizochytrium sp. S31 (ATCC No. 20888) were picked from solidF-1 medium and placed into 50 ml of M-5 medium. This medium consists of(on a per liter basis): yeast extract, 1 g; NaCl, 25 g; MgSO₄,·7H₂ O, 5g; KCl, 1 g; CaCl₂, 200 mg; glucose, 5 g; glutamate, 5 g; KH₂ PO₄, 1 g;PII metals, 5 ml; A-vitamins solution, 1 ml ; and antibiotic solution, 1ml. The pH of the solution was adjusted to 7.0 and the solution wasfilter sterilized. Sterile solutions of corn steep liquor (4 g/40 ml; pH7.0) and yeast extract (1g /40 ml; pH 7.0) were prepared. To one set ofM-5 medium flasks, the following amount of yeast extract solution wasadded: 1) 2 ml; 2) 1.5 ml; 3) 1 ml ; 4) 0.5 ml; and 5) 0.25 ml. Toanother set of M-5 medium flasks the yeast extract and corn steep liquorsolutions were added at the following levels: 1) 2 ml yeast extract; 2)1.5 ml yeast extract and 0.5 ml corn steep liquor; 3) 1.0 ml yeastextract and 1.0 ml corn steep liquor; 4) 0.5 ml yeast extract and 1.5 mlcorn steep liquor; and 5) 2 ml corn steep liquor. One ml of the culturein F-1 medium was used to inoculate each flask. They were placed on arotary shaker at 27° C. for 48 hr. The cells were harvested bycentrifugation and the yield of cells (as ash-free dry weight) wasdetermined. The results are shown in Table 1. The results indicate theaddition of yeast extract up to 0.8 g/l of medium can increase the yieldof cells. However, addition of corn steep liquor is even more effectiveand results in twice the yield of treatments with added yeast extract.This is very advantageous for the economic production of cells as cornsteep liquor is much less expensive than yeast extract.

                  TABLE 1                                                         ______________________________________                                        Treatment                                                                     (Amount Nutrient     Ash-Free Dry Weight                                      Supplement Added)    (mg/l)                                                   ______________________________________                                        2.0 ml yeast ext.        4000                                                 1.5 ml yeast ext.        4420                                                 1.0 ml yeast ext.        4300                                                 0.5 ml yeast ext.        2780                                                 0.25 ml                                                                              yeast ext.        2700                                                 2.0 ml yeast ext.        4420                                                 1.5 ml yeast ext. + 0.5 ml CSL*                                                                        6560                                                 1.0 ml yeast ext. + 1.0 ml CSL                                                                         6640                                                 0.5 ml yeast ext. + 1.5 ml CSL                                                                         7200                                                 2.0 ml CSL               7590                                                 ______________________________________                                         *CSL = corn steep liquor                                                 

Example 4

Enhanced HUFA Content of Strains Isolated by Method in Example 1Compared to ATCC Strains (Previously Known Strains)

A battery of 151 newly isolated strains, selected according to themethod described in Example 1, were sampled in late exponential phasegrowth and quantitatively analyzed for HUFA content by gas-liquidchromatography. All strains were grown either in M1 medium or liquid FFMmedium, whichever gave highest yield of cells. M1 medium has the samecomposition as M5 medium, except that the concentrations of glucose andglutamate are 1 g/l. Additionally, five previously isolatedThraustochytrium or Schizochytrium species were obtained from theAmerican Type Culture Collection, representing all the strains whichcould be obtained in viable form from the collection. These strainswere: T. aureum (ATCC No. 28211), T. aureum (ATCC No. 34304), T. roseurn(ATCC No. 28210), T. straiturn (ATCC No. 34473) and S. aggregatum (ATCCNo. 28209). The strains all exhibited abbreviated growth in conventionalmedia, and generally showed improved growth in media of the presentinvention, including M5 medium and FFM medium. The fatty acid productionof each of the known strains was measured as described, based upon theimproved growth of the strains in media of the invention.

Fatty acid peaks were identified by the use of pure compounds of knownstructure. Quantitation, in terms of percent by weight of total fattyacids, was carried out by integrating the chromatographic peaks.Compounds identified were: palmitic acid (C16:0), C20:4n-6 and C22:1(which were not resolved separately by the system employed), C20:5n-3,C22:5n-6, C22:5n-3, and C22:6n-3. The remainder, usually lower molecularweight fatty acids, were included in the combined category of "otherfatty acids." Total omega-3 fatty acids were calculated as the sum of20:5n-3, 22:5n-3 and 22:6n-3. Total omega-6 fatty acids were calculatedas the sum of the 20:4/22:1 peak and the 22:5n-6 peak.

The results are shown in Tables 2-3 and illustrated in FIGS. 1-3. FromTable 2 it can be seen that large numbers of strains can be isolated bythe method of the invention, and that large numbers of strainsoutperform the previously known strains by several important criteria.For example, 102 strains produced at least 7.8% by weight of total fattyacids C20:5w3, a higher percentage of that fatty acid than anypreviously known strain. Strains 23B (ATCC No. 20892) and 12B (ATCC No.20890) are examples of such strains. Thirty (30) strains of theinvention produced at least 68% by weight of total fatty acids asomega-3 fatty acids, more than any previously known strain. Strain 23B(ATCC No. 20892) is an example of such strains. Seventy-six (76) strainsof the invention yielded not more than 10% by weight of total fattyacids as omega-6 fatty acids, considered undesirable components of thehuman diet, lower than any previously known strain. Strains 23B (ATCCNo. 20892) and B (ATCC No. 20890) are examples of such strains. Inaddition, there are 35 strains of the invention that produce more than25% by weight of total fatty acids as omega-6 fatty acids, more than anypreviously known strain. While such strains may have a more narrow rangeof uses for dietary purposes, they are useful as feedstock for chemicalsynthesis of eicosanoids starting from omega-6 fatty acids.

In addition, the data reveal many strains of the invention which producea high proportion of total omega-3 fatty acids as C22:6n-3. In Table 3,48 of the strains shown in Table 2 were compared to the previously knownstrains, showing each of C20:5n-3, C22:5n-3 and C22:6n-3 as percent byweight of total omega-3 content. Fifteen strains had at least 94% byweight of total omega-3 fatty acids as C22:6n-3, more than anypreviously known strain. Strain S8 (ATCC No. 20889) was an example ofsuch strains. Eighteen strains had at least 28% by weight of totalomega-3 fatty acids as C20:5n-3, more than any previously known strain.Strain 12B (ATCC No. 20890) was an example of such strains.

                                      TABLE 2                                     __________________________________________________________________________    LIST OF STRAINS AND COMPOSITIONS UNDER STANDARD SCREENING CONDITIONS          PER CENT OF TOTAL FATTY ACIDS      Total                                                                              Total                                 C16:0                                                                             C20:4w6                                                                            C20:5w3                                                                            C22:5w6                                                                            C22:5w3                                                                            C22:6w3                                                                            Other FA                                                                            Omega 3                                                                            Omega 6                                                                            Strain                           __________________________________________________________________________    30.4%                                                                             2.8% 6.6% 3.2% 0.2% 0.3% 40.5% 15.1%                                                                              6.0% 21                               22.9%                                                                             0.4% 2.3% 15.5%                                                                              0.5% 47.0%                                                                              11.5% 49.7%                                                                              15.9%                                                                              ATCC20889                        14.9%                                                                             6.5% 12.0%                                                                              11.8%                                                                              0.4% 49.7%                                                                              4.7%  62.1%                                                                              18.3%                                                                              U40-2                            40.3%                                                                             1.7% 3.8% 8.6% 0.0% 8.2% 37.4% 12.0%                                                                              10.2%                                                                              21B                              20.7%                                                                             0.4% 7.8% 0.0% 0.0% 1.1% 70.1% 8.9% 0.4% BGl                              26.0%                                                                             5.7% 1.5% 9.7% 0.7% 9.7% 46.7% 11.9%                                                                              15.4%                                                                              5GA                              16.4%                                                                             1.4% 10.0%                                                                              1.9% 2.2% 46.4%                                                                              21.8% 58.6%                                                                              3.3% 11A-1                            23.7%                                                                             3.3% 10.5%                                                                              1.9% 1.8% 29.9%                                                                              28.9% 42.2%                                                                              5.2% 4A-1                             18.7%                                                                             6.9% 9.2% 11.9%                                                                              3.2% 25.2%                                                                              24.9% 37.5%                                                                              18.8%                                                                              17B                              15.4%                                                                             4.2% 7.3% 9.5% 0.9% 51.2%                                                                              11.6% 59.3%                                                                              13.7%                                                                              ATCC20891                        22.3%                                                                             3.9% 7.6% 23.5%                                                                              0.5% 22.1%                                                                              20.2% 30.2%                                                                              27.4%                                                                              544                              14.4%                                                                             2.3% 15.0%                                                                              18.4%                                                                              0.7% 43.0%                                                                              5.5%  59.4%                                                                              20.7%                                                                              U30                              22.1%                                                                             7.8% 3.1% 12.7%                                                                              1.0% 14.9%                                                                              38.3% 19.0%                                                                              20.5%                                                                              59A                              18.1%                                                                             2.3% 6.9% 9.1% 0.8% 52.2%                                                                              10.6% 59.9%                                                                              11.4%                                                                              U37-2                            15.8%                                                                             3.9% 8.8% 11.6%                                                                              1.2% 53.3%                                                                              5.5%  63.3%                                                                              15.5%                                                                              550W                             23.7%                                                                             3.8% 6.3% 6.9% 0.6% 43.0%                                                                              15.6% 50.0%                                                                              10.7%                                                                              ATCC20891                        10.0%                                                                             0.0% 0.0% 0.0% 0.0% 0.0% 90.0% 0.0% 0.0% UX                               16.6%                                                                             6.3% 11.9%                                                                              13.3%                                                                              1.7% 43.0%                                                                              7.3%  56.6%                                                                              19.5%                                                                              LW9                              17.3%                                                                             2.3% 8.4% 11.4%                                                                              0.7% 53.6%                                                                              6.5%  62.6%                                                                              13.6%                                                                              C32-2                            23.0%                                                                             1.2% 6.4% 2.5% 1.9% 34.4%                                                                              29.8% 42.6%                                                                              3.7% 5A-1                             17.1%                                                                             5.2% 11.1%                                                                              7.6% 2.2% 27.2%                                                                              29.6% 40.4%                                                                              12.9%                                                                              BG1                              25.4%                                                                             2.2% 9.6% 7.0% 1.1% 46.0%                                                                              8.8%  56.7%                                                                              9.1% U3                               16.9%                                                                             12.0%                                                                              6.6% 16.7%                                                                              0.4% 25.1%                                                                              22.8% 32.1%                                                                              28.2%                                                                              55B                              26.3%                                                                             2.6% 8.6% 2.0% 2.5% 32.4%                                                                              25.5% 43.5%                                                                              4.6% 18A                              19.4%                                                                             0.3% 9.8% 0.0% 0.3% 38.4%                                                                              31.7% 48.6%                                                                              0.3% 32B                              16.0%                                                                             16.7%                                                                              8.6% 18.4%                                                                              0.0% 22.5%                                                                              17.7% 31.1%                                                                              35.1%                                                                              56B                              18.6%                                                                             7.7% 11.4%                                                                              3.6% 4.3% 31.7%                                                                              22.7% 47.4%                                                                              11.2%                                                                              SX2                              17.8%                                                                             4.4% 16.2%                                                                              6.4% 3.7% 33.6%                                                                              17.8% 53.5%                                                                              10.9%                                                                              53B                              16.0%                                                                             2.7% 13.8%                                                                              20.5%                                                                              1.4% 33.3%                                                                              5.5%  54.4%                                                                              23.3%                                                                              S49                              20.8%                                                                             8.0% 8.9% 6.4% 1.7% 33.9%                                                                              20.3% 44.5%                                                                              14.4%                                                                              S3                               14.0%                                                                             0.3% 3.7% 3.9% 0.0% 69.9%                                                                              7.4%  73.6%                                                                              4.2% 3A-1                             28.1%                                                                             5.2% 12.7%                                                                              3.2% 0.9% 20.9%                                                                              29.0% 34.5%                                                                              0.4% 15A                              20.9%                                                                             0.7% 0.5% 1.0% 0.0% 35.8%                                                                              33.0% 44.3%                                                                              1.7% 9A-1                             15.7%                                                                             10.2%                                                                              8.8% 13.4%                                                                              1.5% 23.9%                                                                              26.3% 34.3%                                                                              23.7%                                                                              51B                              16.2%                                                                             11.2%                                                                              7.8% 16.4%                                                                              1.5% 20.4%                                                                              26.5% 29.7%                                                                              27.6%                                                                              8A-1                             20.5%                                                                             5.5% 8.6% 4.8% 2.7% 20.7%                                                                              29.2% 40.0%                                                                              10.3%                                                                              13A-1                            16.1%                                                                             13.6%                                                                              11.1%                                                                              16.0%                                                                              0.0% 28.4%                                                                              14.8% 39.4%                                                                              29.6%                                                                              24B-2                            16.9%                                                                             7.3% 16.4%                                                                              6.1% 0.0% 40.8%                                                                              12.4% 57.2%                                                                              13.4%                                                                              24B-1                            16.2%                                                                             0.0% 10.9%                                                                              1.0% 0.0% 56.5%                                                                              15.5% 67.4%                                                                              1.0% 3B                               17.0%                                                                             0.0% 5.0% 2.3% 0.0% 73.4%                                                                              2.3%  78.3%                                                                              2.3% SBG5                             20.8%                                                                             4.5% 5.8% 3.8% 1.0% 22.7%                                                                              41.3% 29.5%                                                                              8.4% 16B                              19.0%                                                                             14.0%                                                                              8.3% 18.9%                                                                              0.7% 23.9%                                                                              15.2% 32.9%                                                                              32.9%                                                                              6A-1                             18.0%                                                                             0.3% 10.1%                                                                              0.0% 0.0% 48.9%                                                                              22.7% 59.0%                                                                              0.3% 33B                              16.7%                                                                             5.5% 14.8%                                                                              8.5% 1.7% 31.8%                                                                              21.0% 48.3%                                                                              13.9%                                                                              B40                              15.0%                                                                             1.0% 11.7%                                                                              2.1% 0.9% 62.3%                                                                              6.9%  74.9%                                                                              3.1% 28A                              17.8%                                                                             18.5%                                                                              8.1% 20.5%                                                                              0.0% 22.1%                                                                              12.9% 30.2%                                                                              39.0%                                                                              43B                              16.9%                                                                             0.0% 3.4% 2.7% 0.0% 61.2%                                                                              15.8% 64.6%                                                                              2.7% 1A-1                             15.6%                                                                             2.7% 11.4%                                                                              10.9%                                                                              0.8% 53.7%                                                                              4.9%  65.9%                                                                              13.6%                                                                              U41-2                            16.5%                                                                             0.7% 3.9% 3.9% 0.0% 68.4%                                                                              6.7%  72.2%                                                                              4.6% 5GB                              14.4%                                                                             0.9% 10.9%                                                                              2.5% 1.0% 66.4%                                                                              3.8%  78.3%                                                                              3.4% 46A                              17.6%                                                                             0.0% 3.3% 0.0% 1.4% 53.2%                                                                              17.1% 57.9%                                                                              0.0% 15A-1                            25.0%                                                                             0.0% 3.3% 0.0% 1.4% 53.2%                                                                              17.1% 57.9%                                                                              0.0% 13A                              16.1%                                                                             13.4%                                                                              9.3% 13.6%                                                                              0.0% 32.3%                                                                              15.3% 41.6%                                                                              27.0%                                                                              37B                              16.5%                                                                             9.1% 13.2%                                                                              6.7% 0.0% 38.9%                                                                              5.6%  52.1%                                                                              15.9%                                                                              43B                              16.1%                                                                             12.4%                                                                              12.0%                                                                              15.7%                                                                              0.8% 30.5%                                                                              12.5% 43.3%                                                                              28.1%                                                                              17B                              13.8%                                                                             0.8% 11.5%                                                                              2.8% 0.0% 67.0%                                                                              4.1%  78.6%                                                                              3.6% 27A                              17.5%                                                                             18.6%                                                                              9.0% 19.5%                                                                              0.0% 21.7%                                                                              13.7% 30.7%                                                                              38.1%                                                                              46B                              21.4%                                                                             1.4% 18.9%                                                                              0.0% 5.0% 43.5%                                                                              9.9%  67.3%                                                                              1.4% ATCC20890                        17.7%                                                                             0.0% 0.6% 4.4% 0.0% 68.2%                                                                              9.1%  68.8%                                                                              4.4% 5A                               17.6%                                                                             16.0%                                                                              9.6% 18.8%                                                                              0.0% 25.6%                                                                              12.4% 35.2%                                                                              34.8%                                                                              2BB-2                            14.0%                                                                             0.9% 13.2%                                                                              1.6% 0.0% 64.7%                                                                              5.5%  77.9%                                                                              2.6% 27B                              19.5%                                                                             2.9% 16.6%                                                                              1.1% 1.6% 30.2%                                                                              28.1% 48.5%                                                                              4.0% 49B                              17.2%                                                                             0.7% 6.8% 2.7% 0.0% 63.0%                                                                              9.6%  69.8%                                                                              3.4% 18B                              14.4%                                                                             3.5% 13.5%                                                                              26.0%                                                                              1.0% 37.2%                                                                              4.4%  51.6%                                                                              29.5%                                                                              S49-2                            16.1%                                                                             2.2% 15.7%                                                                              21.6%                                                                              0.0% 36.7%                                                                              7.8%  52.4%                                                                              23.7%                                                                              20B                              17.3%                                                                             4.7% 14.3%                                                                              7.2% 2.9% 30.2%                                                                              23.5% 47.3%                                                                              11.9%                                                                              8B                               11.5%                                                                             3.3% 11.3%                                                                              6.5% 1.1% 59.9%                                                                              6.5%  72.2%                                                                              9.8% 13B                              16.6%                                                                             0.7% 10.7%                                                                              1.6% 0.0% 59.7%                                                                              10.8% 70.4%                                                                              2.2% 26A                              16.1%                                                                             3.3% 13.5%                                                                              23.8%                                                                              0.0% 38.7%                                                                              4.7%  52.2%                                                                              27.1%                                                                              S42                              15.6%                                                                             0.6% 12.1%                                                                              0.0% 0.0% 60.2%                                                                              11.5% 72.3%                                                                              0.6% 35B                              19.5%                                                                             0.0% 1.4% 3.4% 0.0% 66.6%                                                                              9.1%  68.0%                                                                              3.4% 42A                              18.9%                                                                             3.5% 12.7%                                                                              25.0%                                                                              0.0% 35.0%                                                                              5.0%  47.6%                                                                              28.5%                                                                              40A                              25.2%                                                                             3.3% 9.3% 21.8%                                                                              0.0% 30.3%                                                                              10.1% 39.6%                                                                              25.1%                                                                              S50C                             17.6%                                                                             11.1%                                                                              13.2%                                                                              14.1%                                                                              1.3% 28.7%                                                                              14.0% 43.2%                                                                              25.2%                                                                              59A                              19.9%                                                                             9.9% 5.5% 1.9% 9.9% 66.8%                                                                              6.0%  72.3%                                                                              1.9% SBG9                             15.4%                                                                             3.1% 13.2%                                                                              26.1%                                                                              0.0% 35.8%                                                                              6.5%  49.1%                                                                              29.1%                                                                              21B                              18.9%                                                                             0.7% 11.6%                                                                              0.0% 0.0% 59.1%                                                                              9.7%  70.7%                                                                              0.7% 2B                               14.1%                                                                             1.1% 12.4%                                                                              2.0% 0.0% 65.2%                                                                              5.2%  77.6%                                                                              3.1% 1B                               22.2%                                                                             16.2%                                                                              6.3% 17.7%                                                                              0.0% 18.1%                                                                              19.5% 24.4%                                                                              33.8%                                                                              55B                              16.0%                                                                             1.0% 4.5% 0.0% 0.0% 69.5%                                                                              9.0%  74.0%                                                                              1.0% 3A                               17.0%                                                                             4.3% 12.4%                                                                              29.8%                                                                              0.0% 34.0%                                                                              2.5%  46.4%                                                                              34.1%                                                                              9B                               15.4%                                                                             4.3% 8.7% 13.2%                                                                              0.0% 53.2%                                                                              5.1%  62.0%                                                                              17.5%                                                                              U24                              14.2%                                                                             3.1% 12.0%                                                                              20.0%                                                                              1.1% 35.2%                                                                              14.3% 48.3%                                                                              23.2%                                                                              U28                              16.8%                                                                             14.6%                                                                              10.1%                                                                              16.0%                                                                              0.6% 27.7%                                                                              14.0% 38.5%                                                                              30.7%                                                                              28B-1                            23.2%                                                                             1.9% 8.3% 1.1% 2.3% 22.7%                                                                              40.4% 33.3%                                                                              3.0% 44B                              24.6%                                                                             15.8%                                                                              8.7% 16.0%                                                                              0.0% 15.3%                                                                              19.6% 24.0%                                                                              31.8%                                                                              54B                              15.5%                                                                             0.0% 1.3% 2.9% 0.0% 72.7%                                                                              7.6%  74.0%                                                                              2.9% 55A                              18.4%                                                                             1.0% 5.0% 3.0% 0.0% 66.2%                                                                              6.4%  71.3%                                                                              3.9% 49A                              18.6%                                                                             15.3%                                                                              9.4% 18.0%                                                                              0.0% 27.3%                                                                              11.4% 36.7%                                                                              33.3%                                                                              51A                              23.5%                                                                             13.1%                                                                              7.3% 17.9%                                                                              0.0% 26.7%                                                                              11.4% 34.0%                                                                              31.0%                                                                              14A-1                            13.3%                                                                             1.1% 14.5%                                                                              0,.9%                                                                              0.0% 64.6%                                                                              5.6%  79.1%                                                                              2.0% 25B                              22.9%                                                                             2.4% 10.3%                                                                              21.5%                                                                              0.0% 26.5%                                                                              16.4% 36.9%                                                                              23.9%                                                                              41A                              16.0%                                                                             1.0% 9.7% 2.7% 0.0% 58.3%                                                                              11.5% 68.0%                                                                              3.7% 24A                              0.4%                                                                              8.5% 14.1%                                                                              10.2%                                                                              2.1% 27.6%                                                                              37.0% 43.8%                                                                              18.8%                                                                              G1A                              30.5%                                                                             0.0% 7.1% 0.0% 0.0% 0.6% 61.8% 7.7% 0.0% BRBG                             10.2%                                                                             14.9%                                                                              0.3% 18.7%                                                                              0.0% 24.4%                                                                              15.5% 32.7%                                                                              33.6%                                                                              17A                              17.4%                                                                             2.0% 9.3% 2.8% 0.0% 55.7%                                                                              12.7% 65.0%                                                                              4.9% 60A                              14.1%                                                                             0.8% 13.0%                                                                              1.2% 0.0% 67.8%                                                                              3.1%  80.8%                                                                              2.0% 26B                              17.8%                                                                             5.0% 6.9% 15.0%                                                                              1.5% 47.4%                                                                              6.4%  55.8%                                                                              20.0%                                                                              ATCC20880                        16.0%                                                                             0.0% 1.8% 2.8% 0.0% 70.8%                                                                              9.4%  72.6%                                                                              2.0% 2A                               24.6%                                                                             0.0% 4.0% 0.8% 0.0% 49.4%                                                                              22.0% 53.4%                                                                              0.0% 44A                              17.4%                                                                             1.8% 0.0% 2.9% 0.0% 55.3%                                                                              23.3% 55.3%                                                                              4.6% 14A                              23.3%                                                                             1.3% 4.6% 0.0% 0.0% 12.6%                                                                              58.4% 17.3%                                                                              1.3% 41B                              19.3%                                                                             0.0% 1.1% 3.8% 0.0% 66.6%                                                                              9.1%  67.8%                                                                              3.8% GGA                              18.6%                                                                             15.6%                                                                              8.3% 17.1%                                                                              1.1% 24.6%                                                                              14.8% 33.9%                                                                              32.7%                                                                              11A                              19.6%                                                                             5.1% 10.1%                                                                              27.2%                                                                              0.0% 27.5%                                                                              10.6% 37.5%                                                                              32.3%                                                                              2%                               15.7%                                                                             2.4% 14.0%                                                                              25.7%                                                                              0.0% 36.7%                                                                              5.4%  50.8%                                                                              20.1%                                                                              33A                              14.6%                                                                             1.5% 13.5%                                                                              0.0% 0.0% 66.0%                                                                              4.3%  79.5%                                                                              1.5% ATCC20892                        __________________________________________________________________________

    __________________________________________________________________________    PRIOR STRAINS                                                                 PER CENT OF TOTAL FATTY ACIDS      Total                                                                              Total                                 C16:0                                                                             C20:4w6                                                                            C20:5w3                                                                            C22:5w6                                                                            C22:5w3                                                                            C22:6w3                                                                            Other FA                                                                            Omega 3                                                                            Omega 6                                                                            Strain                           __________________________________________________________________________    15.7%                                                                             3.9% 3.7% 0.1% 0.0% 55.1%                                                                              13.5% 58.8%                                                                              12.0%                                                                              ATCC34304                        28.2%                                                                             1.6% 6.9% 11.4%                                                                              0.0% 17.8%                                                                              34.1% 24.7%                                                                              12.9%                                                                              ATCC24473                        15.2%                                                                             2.9% 7.7% 9.8% 0.6% 54.6%                                                                              9.2%  62.9%                                                                              12.7%                                                                              ATCC28211                        23.2%                                                                             10.7%                                                                              4.3% 12.6%                                                                              1.5% 20.6%                                                                              27.0% 26.4%                                                                              23.4%                                                                              ATCC28209                        13.2%                                                                             6.3% 6.9% 4.3% 0.0% 60.1%                                                                              9.1%  67.0%                                                                              10.6%                                                                              ATCC28210                        __________________________________________________________________________

                  TABLE 3                                                         ______________________________________                                        COMPOSITION OF OMEGA 3 FATTY ACID FRACTION                                    EPA        DPA      DHA                                                       C20:5w3    C22:5w3  C22:6w3      Strain                                       ______________________________________                                        44.0%      1.1%     54.9%        21                                           4.6%       0.9%     94.5%        ATCC20889                                    19.3%      0.7%     80.0%        U40-2                                        31.9%      0.0%     68.1%        21B                                          87.9%      0.0%     12.1%        BRBG1                                        12.5%      6.1%     81.5%        56A                                          17.0%      3.7%     79.3%        11A-1                                        24.9%      4.3%     70.8%        4A-1                                         24.4%      8.4%     67.2%        17B                                          12.2%      1.5%     86.3%        ATCC20891                                    25.1%      1.7%     73.2%        S44                                          25.2%      1.1%     73.7%        U30                                          16.2%      5.4%     78.4%        59A                                          11.5%      1.4%     87.1%        U37-2                                        14.0%      1.9%     84.2%        550W                                         12.7%      1.3%     86.0%        ATCC20891                                    --         --       --           U%                                           21.0%      2.9%     76.1%        LWH9                                         13.4%      1.0%     85.6%        C32-2                                        15.0%      4.3%     80.7%        5A-1                                         27.4%      5.4%     67.2%        BRBG1                                        17.0%      1.9%     81.1%        U3                                           20.5%      1.3%     78.2%        55B                                          19.0%      0.7%     79.2%        32B                                          27.8%      0.0%     72.2%        56B                                          24.1%      9.1%     66.9%        S%2                                          30.3%      6.9%     62.8%        53B                                          25.3%      2.5%     72.2%        S49                                          19.9%      3.8%     76.3%        S3                                           5.0%       0.0%     95.0%        3A-1                                         36.9%      2.6%     60.5%        15A                                          19.3%      0.0%     00.7%        9A-1                                         25.8%      4.4%     69.8%        51B                                          26.3%      5.0%     68.7%        8A-1                                         21.6%      6.7%     71.7%        13A-1                                        28.0%      0.0%     72.0%        24B-2                                        28.7%      0.0%     71.3%        24B-1                                        16.2%      0.0%     83.8%        3B                                           6.3%       0.0%     93.7%        SBG5                                         19.7%      3.3%     77.0%        16B                                          25.7%      2.1%     72.6%        6A-1                                         17.1%      0.0%     82.9%        33B                                          30.5%      3.6%     65.9%        B40                                          15.6%      1.2%     83.1%        28A                                          26.8%      0.0%     73.2%        43B                                          5.2%       0.0%     94.8%        1A-1                                         17.4%      1.2%     81.5%        U41-2                                        5.4%       0.0%     94.6%        56B                                          13.9%      1.3%     84.8%        46A                                          3.5%       0.0%     96.5%        15A-1                                        5.8%       2.4%     91.8%        13A                                          22.3%      0.0%     77.7%        37B                                          25.4%      0.0%     74.6%        43B                                          27.7%      1.9%     70.3%        17B                                          14.7%      0.0%     85.3%        27/A                                         29.2%      0.0%     70.8%        46B                                          28.0%      7.5%     64.5%        ATCC20890                                    0.9%       0.0%     99.1%        5A                                           27.3%      0.0%     72.7%        28B-2                                        16.9%      0.0%     83.1%        27B                                          34.3%      3.4%     62.3%        49B                                          9.7%       0.0%     90.3%        18B                                          26.1%      1.9%     71.9%        S49-2                                        29.9%      0.0%     70.1%        20B                                          30.1%      6.2%     63.7%        8B                                           15.6%      1.5%     82.9%        13B                                          15.2%      0.0%     84.8         26A                                          25.9%      0.0%     74.1%        S42                                          16.7%      0.0%     83.3%        35B                                          2.1%       0.0%     97.9%        42A                                          26.6%      0.0%     73.4%        40A                                          23.4%      0.0%     76.6%        S50C                                         30.6%      2.9%     66.4%        59A                                          7.6%       0.0%     92.4%        SBG9                                         27.0%      0.0%     73.0%        21B                                          16.4%      0.0%     83.6%        2B                                           15.9%      0.0%     84.1%        1B                                           25.9%      0.0%     74.1%        55B                                          6.0%       0.0%     94.0%        3A                                           26.7%      0.0%     73.3%        9B                                           14.1%      0.0%     85.9%        U24                                          24.9%      2.2%     72.9%        U28                                          26.4%      1.5%     72.1%        28B-1                                        24.8%      6.9%     68.3%        44B                                          36.4%      0.0%     63.6%        54B                                          1.8%       0.0%     90.2%        55A                                          7.1%       0.0%     92.9%        49A                                          25.6%      0.0%     74.4%        51A                                          21.5%      0.0%     78.5%        14A-1                                        18.4%      0.0%     81.6%        25B                                          28.1%      0.0%     71.9%        41A                                          14.3%      0.0%     85.7%        24A                                          32.3%      4.8%     63.0%        61A                                          91.6%      0.0%     8.4%         BRBG                                         25.5%      0.0%     74.5%        17A                                          14.4%      0.0%     85.6%        60A                                          16.1%      0.0%     83.9%        26B                                          12.4%      2.7%     84.9%        ATCC20888                                    2.5%       0.0%     97.5%        2A                                           7.5%       0.0%     92.5%        44A                                          0.0%       0.0%     100.0%       14A                                          26.7%      0.0%     73.3%        41B                                          1.7%       0.0%     98.3%        66A                                          24.5%      3.1%     72.4%        11A                                          26.8%      0.0%     73.2%        2%                                           27.6%      0.0%     72.4%        33A                                          17.0%      0.0%     83.0%        ATCC20892                                    ______________________________________                                    

    ______________________________________                                        PRIOR STRAINS                                                                 EPA        DPA      DHA                                                       C20:5w3    C22:5w3  C22:6w3      Strain                                       ______________________________________                                        6.4%       0.0%     93.6%        ATCC34304                                    27.9%      0.0%     72.1%        ATCC24473                                    12.2%      1.0%     86.8%        ATCC28211                                    16.4%      5.6%     78.1%        ATCC28209                                    10.3%      0.0%     89.7%        ATCC28210                                    ______________________________________                                    

FIG. 1 illustrates the set of strains, isolated by the method in Example1, that have more than 67% omega-3 fatty acids (as % of total fattyacids) and less than 10.6% omega-6 fatty acids (as % of total fattyacids). All of the previously known strains had less than 67% omega-3fatty acids (as % of total fatty acids) and greater than 10.6% omega-6(as % of total fatty acids).

FIG. 2 illustrates the set of strains, isolated by the method in Example1, that have more than 67% omega-3 fatty acids (as % of total fattyacids) and greater than 7.5% C20:5n-3 (as % of total fatty acids). Allof the previously known strains had less than 67% omega-3 fatty acids(as % of total fatty acids) and less than 7.8% C20:5n-3 (as % of totalfatty acids ).

Example 5

Enhanced Growth Rates of Strains Isolated by Method in Example 1Compared to ATCC Strains (Previously Known Strains)

Cells of Schizochytrium sp. S31 (ATCC No. 20888), Schizochytrium sp. S8(ATCC No. 20889), Thraustochytrium sp. S42, Thraustochytrium sp. U42-2,Thraustochytrium sp. S42 and U30, (all isolated by the method ofExample 1) and Thraustochytrium aureum (ATCC #28211) and Schizochytriumaggregatum (ATCC #28209) (previously known strains) were picked fromsolid F-1 medium and placed into 50 ml of M-5 medium. The pH of thesolution was adjusted to 7.0 and the solution was filter sterilized.After three days of growth on an orbital shaker (200 rpm, 27° C.), 1-2ml of each culture was transferred to another flask of M-5 medium andplaced on the shaker for 2 days. The cultures (1-2 ml) were thentransferred to another flask of M-5 medium and placed on the shaker for1 day. This process ensured that all cultures were in the exponentialphase of growth. These later cultures were then used to inoculate two250 ml flasks of M-5 medium for each strain. These flasks were thanplaced on shakers at 25° C. and 30° C., and changes in their opticaldensity were monitored on a Beckman DB-G spectrophotometer (660 nm, 1 cmpath length). Optical density readings were taken at the followingtimes: 0, 6, 10, 14, 17.25, 20.25 and 22.75 hours. Exponential growthrates (doublings/day) were then calculated from the optical density databy the method of Sorokin (1973). The results are presented in Table 4and illustrated (normalized to the growth of strain U30 at 25° C.) inFIG. 4. The data indicate that the strains isolated by the method inExample 1 have much higher growth rates than the previously known ATCCstrains at both 25° C. and 30° C., even under the optimized phosphatelevels essential for continuous growth. Strains of Thraustochytrialesisolated from cold Antarctic waters have not been shown to grow at 30°C.

                  TABLE 4                                                         ______________________________________                                        Exponential Growth Rate (doublings/day)                                       Strain              25° C.                                                                         30° C.                                     ______________________________________                                        S31* (ATCC No. 20888)                                                                             8.5     9.4                                               U40-2*              5.8     6.0                                               S8* (ATCC No. 20889)                                                                              7.1     8.8                                               S42*                6.6     8.3                                               U30*                5.5     7.3                                               28209**             4.6     5.0                                               28210**             3.5     4.5                                               28211**             4.2     5.7                                               34304**             2.7     3.7                                               24473**             4.6     5.3                                               ______________________________________                                         *strain isolated by method in Example 1                                       **previously known ATCC strain                                           

Example 6

Enhanced Production Characteristics (Growth and Lipid Induction) ofStrains Isolated by Method in Example 1 Compared to ATCC Strains (PriorArt Strains)

Cells of Schizochytrium sp. S31 (ATCC No. 20888), Schizochytrium sp. S8(ATCC No. 20889) (both isolated by the method of Example 1) andThraustochytrium aureum (ATCC #28211) and Schizochytrium aggregatum(ATCC #28209) (prior art strains) were picked from solid F-1 medium andplaced into 50 ml of M-5 medium (see Example 3). The pH of the solutionwas adjusted to 7.0 and the solution was filter sterilized. After threedays of growth on an orbital shaker (200 rpm, 27° C.), 1-2 ml of eachculture was transferred to another flask of M-5 medium and placed on theshaker for 2 days. The ash-free dry weights for each of these cultureswere then quickly determined and then 3.29 mg of each culture waspipetted into two 250 ml erlenmeyer flasks containing 50 ml of M-5medium. These flasks were placed on a rotary shaker (200 rpm, 27° C.).After 24 hours 20 ml portions of each culture were then centrifuged, thesupernatants discarded, and the cells transferred to 250 ml erlenmeyerflasks containing 50 ml of M-5 medium without any glutamate (N-source).The flasks were placed back on the shaker, and after another 12 hoursthey were sampled to determine ash-free dry weights and quantify fattyacid contents by the method of Lepage and Roy (1984). The results areillustrated (normalized to the yields of ATCC No. 28211, previouslyknown strain) in FIG. 5. The results indicate that the strains isolatedby the method of Example 1 produced 2-3 times as much ash-free dryweight in the same period of time, under a combination of exponentialgrowth and nitrogen limitation (for lipid induction) as the prior artATCC strains. In addition, higher yields of total fatty acids andomega-3 fatty acids were obtained from strains of the present inventionwith strains S31 (ATCC No. 20888) producing 3-4 times as much omega-3fatty acids as the prior art ATCC strains.

Example 7

Enhanced Lower Salinity Tolerance and Fatty Acid Production by StrainsIsolated by Method in Example 1

Strains of 4 species of Thraustochytrids, Schizochytrium sp. S31 (ATCCNo. 20888) and Thraustochytrium sp. U42-2 (ATCC No. 20891) (bothisolated and screened by the method of Example 1), and S. aggregatum(ATCC 28209) and T. aureum (ATCC 28210) (obtained from the American TypeCulture Collection) were picked from solid F-1 medium and incubated for3-4 days at 27° C. on a rotary shaker (200 rpm). A range of differingsalinity medium was prepared by making the following dilutions of Mmedium salts (NaCl, 25 g/l; MgSO₄ ·7H₂ O, 5 g/l; KCl, 1g/l; CaCl₂, 200mg/l: 1) 100% (w/v M medium salts; 2) 80% (v/v) M medium, 20%(v/v)distilled water; 3) 60% (v/v) M medium, 40% (v/v) distilled water;4) (v/v) M medium, 60% (v/v)distilled water; 5) 20% (v/v) M medium, 80%distilled water; 6) 15% (v/v) M medium, 85% (v/v) distilled water; 7)10% (v/v) M medium, 90% (v/v) distilled water; 8) 7% (v/v)M medium, 93%(v/v) distilled water; 9) 3% (v/v) M medium, 97% (v/v) distilled water;10) 1.5% (v/v) M medium, 98.5% (v/v) distilled water. The followingnutrients were added to the treatments (per liter): glucose, 5 g;glutamate, 5 g; yeast ext., 1 g; (NH₄)₂ SO₄, 200 mg; NaHCO3, 200 mg; PIImetals, 5 ml; A-vitamins solution, 1 ml ; and antibiotics solution, 2ml. Fifty ml of each of these treatments were inoculated with 1 ml ofthe cells growing in the F-1 medium. These cultures were placed on anorbital shaker (200 rpm) and maintained at 27° C. for 48 hr. The cellswere harvested by centrifugation and total fatty acids determined by gaschromatography. The results are illustrated in FIG. 6. Thraustochytriumsp. U42-2 (ATCC No. 20891) isolated by the method of Example 1 can yieldalmost twice the amount of fatty acids produced by T. aureum (ATCC28211) and over 8 times the amount of fatty acids produced by S.aggregatum (ATCC 28209). Additionally, U42-2 appears to have a widersalinity tolerance at the upper end of the salinity range evaluated.Schizochytrium sp. S31 (ATCC No. 20888), also isolated by the method inExample 1, exhibited both a high fatty acid yield (2.5 to 10 times thatof the previously known ATCC strains) and a much wider range of salinitytolerance than the ATCC strains. Additionally, Schizochytrium sp. S31(ATCC No. 20888) grows best at very low salinities. This propertyprovides a strong economic advantage when considering commercialproduction, both because of the corrosive effects of saline waters onmetal reactors, and because of problems associated with the disposal ofsaline waters.

Example 8

Cultivation/Low Salinity

Fifty ml of M/10-5 culture media in a 250 ml erlenmeyer flask wasinoculated with a colony of Schizochytrium sp. S31 (ATCC No. 20888)picked from an agar slant. The M/10-5 media contains: 1000 ml deionizedwater, 2.5 g NaCl, 0.5 g MgSO₄ ·7H₂ O, 0.1 g KCl, 0.02 g CaCl₂, 1.0 gKH₂ PO₄, 1.0 g yeast extract, 5.0 g glucose, 5.0 g glutamic acids, 0.2 gNaHCO₃, 5 ml PII trace metals, 2 ml vitamin mix, and 2 ml antibioticmix. The culture was incubated at 30° C. on a rotary shaker (200 rpm).After 2 days the culture was at a moderate density and actively growing.20 ml of this actively growing culture was used to inoculate a 2 literfermenter containing 1700 ml of the same culture media except theconcentration of the glucose and glutamate had been increased to 40 g/l(M/10-40 media). The fermenter was maintained at 30° C., with aerationat 1 vol/vol/min, and mixing at 300 rpm. After 48 hr, the concentrationof cells in the fermenter was 21.7 g/l. The cells were harvested bycentrifugation, lyophilized, and stored under N₂.

The total fatty acid content and omega-3 fatty acid content wasdetermined by gas chromatography. The total fatty acid content of thefinal product was 39.0% ash-free dry weight. The omega-3 HUFA content(C20:5n-3, C22:5n-3 and C22:6n-3) of the microbial product was 25.6% ofthe total fatty acid content. The ash content of the sample was 7.0%.

Example 9

Diversity of Fatty Acid Content

Growth and gas chromatographic analysis of fatty acid production byvarious strains as described in Example 4 revealed differences in fattyacid diversity. Strains of the present invention synthesized fewerdifferent fatty acids than previously available strains. Lower diversityof fatty acids is advantageous in fatty acid purification since thereare fewer impurities to be separated. For food supplement purposes,fewer different fatty acids is advantageous because the likelihood ofingesting unwanted fatty acids is reduced. Table 5 shows the number ofdifferent HUFAs present, at concentrations greater than 1% by weight oftotal fatty acids for previously known strains, designated by ATCCnumber and various strains of the present invention.

                  TABLE 5                                                         ______________________________________                                                    No. of Different Fatty                                                        Acids at 1% or Greater %                                          Strain      of Total Fatty Acids                                              ______________________________________                                        34304**     8                                                                 28211**     8                                                                 24473**     10                                                                28209**     13                                                                28210**     8                                                                 S31*        5                                                                 S8*         6                                                                 79B*        6                                                                 ______________________________________                                         *strain isolated by the method in Example 1                                   **previously known ATCC strain                                           

Example 10

Recovery

Fifty ml of M5 culture media in a 250 ml erlenmeyer flask was inoculatedwith a colony of Schizochytrium sp. S31 (ATCC No. 20888) picked from anagar slant. The culture was incubated at 30° C. on a rotary shaker (200rpm). After 2 days the culture was at a moderate density and activelygrowing. 20 ml of this actively growing culture was used to inoculate a1 liter fermenter containing 1000 ml of the same culture media exceptthe concentration of the glucose and glutamate had been increased to 40g/l (M20 media). The fermenter was maintained at 30° C. and pH 7.4, withaeration at 1 vol/min, and mixing at 400 rpm. After 48 hr, theconcentration of the cells in the fermenter was 18.5 g/l. Aeration andmixing in the fermenter was turned off. Within 2-4 minutes, the cellsflocculated and settled in the bottom 250 ml of the fermenter. Thisconcentrated zone of cells had a cell concentration of 72 g/l. This zoneof cells can be siphoned from the fermenter, and: (1) transferred toanother reactor for a period of nitrogen limitation (e.g., combining thehighly concentrated production of several fermenters); or (2) harvesteddirectly by centrifugation or filtration. By preconcentrating the cellsin this manner, 60-80% less water has to be processed to recover thecells.

Example 11

Utilization of a Variety of Carbon and Nitrogen Sources

Fifty ml of M5 culture media in a 250 ml erlenmeyer flask was inoculatedwith a colony of Schizochytrium sp. S31 (ATCC No. 20888) orThraustochytrium sp. U42-2 (ATCC No. 20891) picked from an agar slant.The M5 media was described in Example 3 except for the addition of 2 mlvitamin mix, and 2 ml antibiotic mix. The culture was incubated at 30°C. on a rotary shaker (200 rpm). After 2 days the culture was at amoderate density and actively growing. This culture was used toinoculate flasks of M5 media with one of the following substituted forthe glucose (at 5 g/l): dextrin, sorbitol, fructose, lactose, maltose,sucrose, corn starch, wheat starch, potato starch, ground corn; or oneof the following substituted for the glutamate (at 5 g/l): gelysate,peptone, tryptone, casein, corn steep liquor, urea, nitrate, ammonium,whey, or corn gluten meal. The cultures were incubated for 48 hours on arotary shaker (200 rpm, 27° C.). The relative culture densities,representing growth on the different organic substrates, are illustratedin Tables 6-7.

                  TABLE 6                                                         ______________________________________                                        Utilization of Nitrogen Sources                                                         Strains                                                                         Thraustochytrium                                                                           Schizochytrium                                                   sp. U42-2    sp. S31                                              N-Source    ATCC No. 20891                                                                             ATCC No. 20888                                       ______________________________________                                        glutamate   +++          +++                                                  gelysate    +++          +++                                                  peptone     ++           ++                                                   tryptone    ++           ++                                                   casein      ++           ++                                                   corn steep  +++          +++                                                  liquor                                                                        urea        +            ++                                                   nitrate     ++           +++                                                  ammonium    +            +++                                                  whey        +++          +++                                                  corn gluten +++          +++                                                  meal                                                                          ______________________________________                                         +++ = high growth                                                             ++ = medium growth                                                            + = low growth                                                                0 = no growth                                                            

                  TABLE 7                                                         ______________________________________                                                  Strains                                                                         Thraustochytrium                                                                           Schizochytrium                                                   sp. U42-2    sp. S31                                              C-Source    ATCC No. 20891                                                                             ATCC No. 20888                                       ______________________________________                                        glucose     +++          +++                                                  dextrin     +++          +++                                                  sorbitol    +            +                                                    fructose    +            +++                                                  lactose     +            +                                                    maltose     +++          +                                                    sucrose     +            +                                                    corn starch +++          +                                                    wheat starch                                                                              +++          +                                                    potato starch                                                                             +++          +                                                    ground corn +++          0                                                    ______________________________________                                         +++ = high growth                                                             ++ = medium growth                                                            + = low growth                                                                0 = no growth                                                            

Example 12

Feeding of Thraustochytrid-Based Feed Supplement to Brine Shrimp toIncrease Their Omega-3 HUFA Content

Cellular biomass of Thraustochytrium sp. 12B (ATCC 20890) was producedin shake flasks in M-5 medium (see Example 3) at 25° C. Cellular biomassof Thraustochytrium sp. S31 (ATCC 20888) was produced in shake flasks inM/10-5 medium (see Example 8) at 27° C. The cells of each strain wereharvested by centrifugation. The pellet was washed once with distilledwater and recentrifuged to produce a 50% solids paste. The resultingpaste was resuspended in sea water and then added to an adult brineshrimp culture as a feed supplement. The brine shrimp had previouslybeen reared on agricultural waste products and as a result their omega-3HUFA content was very low, only 1.3-2.3% of total fatty acids(wild-caught brine shrimp have an average omega-3 HUFA content of 6-8%total fatty acids). The brine shrimp (2-3/ml) were held in a 1 literbeaker filled with sea water and an airstone was utilized to aerate andmix the culture. After addition of the feed supplement, samples of thebrine shrimp were periodically harvested, washed, and their fatty acidcontent determined by gas chromatography. The results are illustrated inFIGS. 7 and 88. When fed the thraustochytrid-based feed supplement as afinishing feed, the omega-3 content of the brine shrimp can be raised tothat of wild-type brine shrimp within 5 hours if fed strain 12B orwithin 11 hours when fed strain S31. The omega-3 HUFA content of thebrine shrimp can be greatly enhanced over that of the wild type if fedthese feed supplements for up to 24 hours. Additionally, these feedsupplements greatly increase the DHA content of the brine shrimp, whichis generally only reported in trace levels in wild-caught brine shrimp.

Example 13

Use of Sodium Sulfate in Culture Medium

This example illustrates that omega-3 production and total fatty acidcontent is not harmed and can be the same or better when using sodiumsulfate instead of sodium chloride as the sodium salt in a fermentationmedium.

Schizochytrium ATCC No. 20888 was grown in medium, pH 7.0, containing2.36 grams of sodium per liter of medium, 1.5-3.0 grams of a nitrogensource per liter of medium, and 3.0 grams of glucose per liter ofmedium. The cells were incubated at 28° C., at 200 rotations per minute,for 48 hours. The results are shown in Table 8.

                  TABLE 8                                                         ______________________________________                                        Effect of Sodium Sulfate Compared With Sodium                                 Chloride on Fatty Acid Content                                                                       total    biomass                                       N source omega-3       fatty acid                                                                             yield                                         (g/L)    (% dwt)       (% dwt)  (g/L)                                         ______________________________________                                        A) Na salt = sodium chloride; N source = sodium glutamate                     3.0      6.0           11.2     1.74                                          2.5      5.8           10.8     1.71                                          2.0      5.8           11.0     1.65                                          1.5      7.5           20.3     1.39                                          B) Na salt = sodium chloride; N source = peptone                              3.0      7.9           21.9     1.34                                          2.5      9.4           27.4     1.21                                          2.0      6.7           28.9     1.18                                          1.5      11.1          42.1     1.16                                          C) Na salt = sodium sulfate; N source = sodium glutamate                      3.0      9.3           31.9     1.34                                          2.5      10.1          38.6     1.35                                          2.0      10.1          41.4     1.30                                          1.5      9.5           43.6     1.26                                          ______________________________________                                    

As seen in Table 8, omega-3 and total fatty acid production when usingsodium sulfate is comparable to or better than when using sodiumchloride as a sodium salt.

Example 14

Production of Schizochytrium in Low Salinity Culture Medium

This Example illustrates the fermentation of Schizochytrium in a lowsalinity culture medium while maintaining high biomass yields and highomega-3 and fatty acid production.

Schizochytrium ATCC No. 20888 was grown in medium, containing 3.33 g/lof peptone as a nitrogen source, 5.0 g/l of glucose as a carbon source,with varying sodium concentrations. The cells were fermented at 30° C.with an inoculum of about 40 mg/L dwt for a period of 48 hours. Thesodium was supplied as sodium chloride. The results of this run areshown in Table 9.

                  TABLE 9                                                         ______________________________________                                        Production of Schizochytrium in Low Salinity                                  Culture Medium                                                                Na            Biomass   Fatty            final                                conc.                                                                              Cl conc. Yield     acids    omega-3 glucose                              g/L  g/L      g/L       % dwt    % dwt   g/L                                  ______________________________________                                        4.88 7.12     1.76 ± 0.60                                                                          35.4 ± 1.0                                                                          10.2 ± 0.6                                                                         0.00                                 3.90 5.70     1.72 ± 0.67                                                                          37.0 ± 0.7                                                                          11.1 ± 0.3                                                                         0.15                                 2.93 4.27     1.70 ± 0.42                                                                          43.0 ± 0.2                                                                          12.1 ± 0.1                                                                         0.22                                 1.95 2.85     1.66 ± 0.57                                                                          29.8 ± 0.7                                                                           9.3 ± 0.1                                                                         1.55                                 0.98 1.42     0.40 ± 0.61                                                                          10.6 ± 2.4                                                                           4.0 ± 1.0                                                                         4.31                                 ______________________________________                                    

As can be seen from the results in Table 9, high biomass yields andproduction of omega-3 fatty acids and total fatty acids can be achievedat sodium concentrations of greater than about 1.0 g/l.

Example 15

Cultivation of Schizochytrium in Medium with Low Chloride Content

This Example illustrates the fermentation of microflora of the presentinvention at minimal chloride concentrations while achieving highbiomass yields based on starting sugar concentration.

Schizochytrium ATCC No. 20888 was cultured in shake flasks at 200 rpmand 28° C. in 50 ml aliquots of the following medium. 1000 ml deionizedwater; 1.2 g Mg SO₄ ·7H₂ O; 0.067 g CaCO₃ ; 3.0 g glucose; 3.0 gmonosodium glutamate; 0.2 g KH₂ PO₄ : 0.4 g yeast extract; 5.0 ml PIImetals, 1.0 vitamin mix; and 0.1 g each of penicillin-G and streptomycinsulfate. The chloride concentration was varied by adding differingamounts of KCl to each treatment. The potassium concentration in all ofthe treatments was held constant by additions of potassium citrate.Sodium concentration was either 2.37 g/l or 4.0 g/l through addition ofsodium sulfate. The results of these fermentations are shown below inTable 10.

                  TABLE 10                                                        ______________________________________                                        Fermentation of Schizochytrium at Minimal Chloride                            Concentrations                                                                              Na 2.37 g/L Na 4.0 g/L                                          Chloride conc.                                                                              Biomass Yield                                                                             Biomass Yield                                       (mg/L)        (mg/L)      (mg/L)                                              ______________________________________                                        0.1            198 ± 21                                                                              158 ± 48                                         7.1            545 ± 120                                                                             394 ± 151                                        15.1           975 ± 21                                                                              758 ± 163                                        30.1          1140 ± 99                                                                              930 ± 64                                         59.1          1713 ± 18                                                                              1650 ± 14                                        119.1         1863 ± 53                                                                              1663 ± 46                                        238.1         1913 ± 11                                                                              1643 ± 39                                        ______________________________________                                    

As can be seen from the results shown in Table 10, high yields ofbiomass per sugar can be achieved at low chloride concentrations. Forexample, at a chloride concentration of greater than 59.1mg/L, yields ofgreater than 50% are achieved.

Example 16

Variation of Sodium sulfate concentration at Low Chloride Concentrations

This Example illustrates the effect of varying sodium sulfateconcentration in a fermentation at low chloride concentration.

Schizochytrium ATC 20888 was cultured in shake flasks at 200 rpm and 28°C. in 50 ml aliquots of the following medium: 1000 ml deionized water;1.2 g MgSO₄ ·7H₂ O; 0.125 g KCl; 0.067 g CaCO₃ ; 3; 3.0 g glucose; 3.0 gmonosodium glutamate; 0.2 g KH₂ PO₄ ; 0.4 g yeast extract; 5.0 ml PIImetals; 1.0 ml vitamin mix; and 0.1 g each of penicillin-G andstreptomycin sulfate. The sodium sulfate concentration was varied in thetreatments from 3.0 g/l to 30.2 g/l. The results of the fermentationruns are shown below in Table 11.

    ______________________________________                                        Variation of Sodium Sulfate Concentration at Low                              Chloride Content                                                              Sodium Sulfate Biomass yield                                                  (g/l)          (g/l)                                                          ______________________________________                                        3.0            0.78                                                           6.0            1.13                                                           9.1            1.72                                                           12.1           1.88                                                           15.1           1.89                                                           22.7           1.91                                                           30.2           1.63                                                           ______________________________________                                    

The results shown in Table 11, illustrate that at a low chlorideconcentration of about 59 g/l,high biomass yields from glucose ofgreater than 50% can be obtained by selection of an appropriate sodiumsulfate concentration.

What is claimed is:
 1. A microfloral biomass comprising a microorganismselected from the group consisting of Thraustochytrium, Schizochytrium,and mixtures thereof, wherein said, microfloral biomass has a cellaggregate size less than about 150 microns in diameter and wherein saidmicrofloral biomass is capable of being produced by a method comprisinggrowing said microorganism and mixtures thereof, in a culture mediumcontaining less than about 3 grams of chloride per liter of said culturemedium, sources of carbon, nitrogen, micronutrients, and a non-chloridesodium salt at a temperature less than about 48° C., and at a pH fromabout pH 5.0 to about pH 11.0.
 2. The microfloral biomass of claim 1,wherein said non-chloride sodium salt is sodium sulfate.
 3. Themicrofloral biomass of claim 1, wherein said microorganism has all ofthe identifying characteristics of a microorganism selected from thegroup consisting of ATCC Nos. 20888, 20889, and mutant strains derivedtherefrom, wherein said microorganism of the microfloral biomass has anomega-3 highly unsaturated fatty acid content of at least about 0.5% dryweight.
 4. The microfloral biomass of claim 1, wherein saidmicroorganism of the microfloral biomass has a sterol content of atleast about 0.1% ash free dry weight.
 5. The microfloral biomass ofclaim 4, wherein said microorganism of the mcirofloral biomass hascholesterol content of at least about 15% of the total sterol content.6. The microfloral biomass of claim 1, wherein said microfloral biomasshas a cell aggregate size less than about 100 microns in diameter. 7.The microfloral biomass of claim 1, wherein said microfloral biomass hasa cell aggregate size less than about 50 microns in diameter.